Leone L, Monteleone M, Gabutti V, Amione C
J Chromatogr. 1985 Mar 15;321(2):407-19. doi: 10.1016/s0021-9673(01)90459-5.
A reversed-phase high-performance liquid chromatographic method for the separation of human haemoglobin chains has been devised. Using a LiChrospher 100 CH-8/2 column and a ternary eluent (acetonitrile-methanol-0.155 M NaCl, pH 2.7) improved resolution was achieved between (delta beta) Lepore, beta A, beta S, alpha, G gamma and A gamma chains within a 60-min linear gradient. The A gamma T chain can also be separated by increasing the gradient time and decreasing the flow-rate. Silanophilic interactions play an important role in the retention mechanism, and NaCl addition was necessary in order to suppress adsorption on free silanols. Increasing the methanol concentration to 10% caused a slight increase in chain retention, probably owing to solvation of the stationary phase. The recovery was 82% and the reproducibility of retention times was as good as +/- 1.5%. Quantitation of chains is likely to be possible by peak area measurement. Owing to its sensitivity, the proposed method may be useful in the diagnosis of haemoglobinopathies and in the study of haemoglobin variants.
已设计出一种用于分离人血红蛋白链的反相高效液相色谱法。使用LiChrospher 100 CH - 8/2柱和三元洗脱液（乙腈 - 甲醇 - 0.155 M NaCl，pH 2.7），在60分钟线性梯度内，（δβ） Lepore、βA、βS、α、Gγ和Aγ链之间实现了更好的分离。通过增加梯度时间和降低流速，也可以分离AγT链。硅醇相互作用在保留机制中起重要作用，添加NaCl是为了抑制在游离硅醇上的吸附。将甲醇浓度提高到10%会导致链保留略有增加，这可能是由于固定相的溶剂化作用。回收率为82%，保留时间的重现性高达±1.5%。通过峰面积测量可能实现链的定量。由于其灵敏度，所提出的方法可能有助于血红蛋白病的诊断和血红蛋白变体的研究。
